The oral cavity is host to many microorganisms comprising bacteria, fungi and viruses. Both acquired and innate host immunity mechanisms prevent and control manifestations of local infection under most physiological conditions. Recent studies have emphasized the importance of oral infections since their effects may not be limited to the integrity of oral hard and soft tissues alone but also represent risks with regard to a variety of systemic diseases. Histatins are basic, small molecular weight, proteins secreted by both the parotid and submandibular/sublingual glands exhibiting direct and indirect antimicrobial activities. The broad and long term objective of this proposal is to understand mechanisms of the salivary host defense system at the molecular level providing opportunities to exploit such information for prevention and therapeutics. A major focus of investigation is the antifungal effect of histatins on the oral pathogen Candida albicans, causing the most frequently occurring oral fungal infection. While a number of molecular and cellular observations have been described the precise mechanism of histatin induced cell death has been elusive. The proposed studies represent a comprehensive and systematic approach for elucidating this mechanism using both quantitative global proteomics and genetics studies which include comparisons of single gene deletions and gene overexpression. These studies promise to be of translational importance considering the paucity of available antifungal agents and the alarming emergence of fungal resistance to available antimycotics. Aim 1 focuses on proteomic changes in C. albicans cells which are associated with histatin susceptibility and resistance. For this purpose protein preparations of subcellular fractions of C. albicans which had been exposed to histatin 5 will be compared to controls over time. For quantitative comparisons three stable isotope methods will be employed. These include isotope coded affinity tag (ICAT), multiplexed isobaric isotope reagent (iTRAQ) and metabolic labeling. Data obtained by mass spectrometry will be subjected to database searches, bioinformatics and statistical analyses to reveal changes in protein expression levels in response to histatin 5 treatment. In Aim 2 the genetic basis for histatin susceptibility will be investigated in a S. cerevisiae model. Histatin susceptibility will be assessed in comprehensive panels of yeast deletion and overexpression mutants using growth inhibition and cell killing assays. In addition, homologues of the functionally important genes in C. albicans will be disrupted by targeted deletion. Integration of the results from both proteomic and genetics approaches will allow us to identify the critical pathways of the histatin killing mechanism. Knowledge of such pathways represents the basis for the development of new second generation antimycotics.